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. 2010 Mar 25;29(9):1499–1509. doi: 10.1038/emboj.2010.46

Figure 6.

Figure 6

USP9x promotes TJ assembly via its activity on EFA6. (A) MDCK cells were transfected with a control siRNA or two distinct siRNA specific for USP9x (#182 and #3432). At 24 h post-transfection, the cells were subjected to a calcium switch and fixed 1 or 3 h after calcium repletion. The samples were co-stained for USP9x (green) and ZO-1 (red). Scale bars, 25 μm. (B) Immunoblots showing USP9x, vsvg-EFA6A and actin levels in tet-off regulated vsvg-EFA6A MDCK cells grown in the absence or presence of doxycyclin (Dox) and transfected with control or USP9x-specific siRNA #182. (C) These cells were analysed for their capacity to form functional TJ. Vsvg-EFA6A MDCK cells transfected with control or USP9x (#182) siRNAs and grown in the absence or presence of doxycycline (+/−Dox) were subjected to a calcium switch and fixed 90 min after calcium repletion and processed for IF analysis. The expression and localization of USP9x (green), ZO-1 (red) and vsvg-EFA6A (blue) were examined. Scale bars, 25 μm. (D) The gain of TJ barrier function was analysed in a calcium switch assay by measuring the TER over time and the paracellular diffusion of TRITC-dextran at 4 h after calcium repletion +/− siUSP9x #182. For TER measurement n=5 and error bars represent the s.e.m. For siControl cells +/−Dox P<0.0025 at all times, for siUSP9x cells +/−Dox P<0.0007 after 4 h, for +Dox cells +/−siUSP9x P<0.0014 after 4 h, for –Dox cells +/− siUSP9x P<0.0012 at all times. For the paracellular diffusion of the TRITC-dextran n=3 and error bars represent s.e.m. For siControl cells +/−Dox P=0.0058, for siUSP9x cells +/−Dox P=0.0049, for +Dox cells +/−siUSP9x P=0.0014, for –Dox cells +/− siUSP9x P=0.0012.